This invention relates to processes for the production of proteins by micro-organisms. Specifically, it relates to the secretion of heterologous proteins by micro-organisms, in particular by Gram-positive bacteria, especially by the bacterial host Bacillus.
It also relates to (the overexpression of) a novel gene encoding a protein involved in the early stages of prokaryotic protein secretion. Specifically, it relates to the overexpression of said gene within a Bacillus host (over)expressing heterologous proteins.
B. subtilis and (closely) related bacilli secrete proteins directly into the growth medium to high concentrations. Secretion as a mode of production of proteins of interest, be it homologous to the host or heterologous to the host, be it of recombinant origin or not, provides several advantages over intracellular production. It for instance facilitates purification of the product, it theoretically will lead to a higher yield, no aggregation of the product will occur, and it gives the possibility for continuous cultivation and production. However, attempts to secrete heterologous proteins from B. subtilis and (closely) related organisms at commercially significant concentrations have, with few exceptions, met with little success.
Nearly all secreted proteins use an amino-terminal protein extension, known as the signalpeptide, which plays a crucial role in the targeting to, and translocation of precursor proteins across the membrane and which is proteolytically removed by a signalpeptidase during or immediately following membrane transfer. The newly synthesized precursor proteins are recognized by specific proteins in the cytoplasm collectively called chaperones. These chaperones prevent polypeptides, destined for translocation, to aggregate or fold prematurely leading to an export incompatible conformation.
For instance, SecB, GroEL/GroES and DnaK/DnaJ are the presently known chaperones in the export pathway of E. coli. For the productive binding of precursor proteins to translocation sites in the cytoplasmic membrane SecA is needed. SecA, a protein of which cytoplasmic, peripheral as well as integral membrane forms have been detected, has an ATPase activity which mediates the initial channelling of precursor proteins into the export pathway.
The SecA subunit acts as a receptor recognizing the leader and mature domains of the preproteins (Lill et al. 1990) as well as the SecB chaperone (Hartl et al. 1990). It has been suggested that SecA penetrates into the membrane, after binding of ATP, and so promotes the coinsertion of the preprotein. After hydrolysis of bound ATP the preprotein is released from the SecA protein (Schiebel et al. 1991). Translocation is completed with the proton motive force as the main driving force and requires members of the integral membrane part of the preprotein translocase complex like SecY, SecE and SecG (p12/Band1). SecD and SecF are also integral membrane proteins and are probably participating in the late steps of protein translocation.
For many years, the protein secretion machinery in prokaryotes has been considered to be independent from the protein secretion system found in higher eukaryotes (Luirink et al, 19920. In mammalians, targeting of secretory proteins to the endoplasmic reticulum (ER) is mediated by the signal recognition particle (SRP), which is a ribonucleoprotein particle composed of one RNA molecule (SRP 7S RNA) and six polypeptides of 9, 14, 19, 54, 68 and 72 kD. The SRP proteins are associated with the RNA as either monomers (SRP19 and SRP54) or heterodimers (SRP9/14 and SRP68/72).
As soon as the signal peptide of secreted and transmembrane proteins has emerged from the ribosome, it is recognized and bound by SRP, which also has affinity for the ribosome. This association slows down the elongation of the polypeptide chain (elongation arrest). When the complex of SRP, nascent polypeptide chain, and ribosome bind to the SRP receptor (SR or docking protein) associated with the ER membrane, the nascent polypeptide chain is displaced from SRP in a GTP-dependent reaction and protein translation is resumed.
The translocation of the polypeptide into the ER takes place co-translationally through a protein pore, the translocon (Gilmore et al. 1993). Thus, the SRP functions both as a cytosolic chaperone preventing premature folding of the preprotein by coupling translation to translocation and as a pilot to guide the preprotein to the SRP receptor complex in the membrane. The 54 kD subunit of SRP (SRP54) binds to the signal peptide when it emerges from the ribosome and therefore seems to have a key function in the SRP-mediated process of protein secretion.
To day more and more data become available indicating that an SRP-mediated export pathway may also function in other organisms. Homologues of mammalian SRP components have been isolated from Yeast (Hann et al. 1989), E. coli (Bernstein et al. 1989, and Rymisch et al. 1989), Mycoplasma mycoides (Samuelsson, 1992) and Bacillus subtilis (Struck et al. 1989, and Honda et al. 1993).
So it is likely that an SRP-mediated pathway functions in prokaryotes in a separate secretory pathway or may form part of the general secretory pathway.
In E. coli members of an SRP-like secretory pathway were identified. These members are Ffh (Fifty four homologue) and a 4.5S RNA molecule which are homologous to the SRP54 and SRP 7S RNA of eukaryotic SRP (Ribes et al. 1990). It is shown that Ffh interacts specifically with the signal sequence of nascent presecretory proteins (Luirink et al. 1992). E. coli protein FtsY, which originally has been implicated in cell division (because its gene is located in an operon together with FtsE and FtsX) displays striking sequence similarity with the subunit of mammalian docking protein. Several observations suggest that FtsY is the functional E. coli homologue of the mammalian SRP receptor (Luirink et al. 1994). Depletion of either FtsY, Ffh or the RNA component of the E. coli SRP affects the export of several secretory proteins.
Also in B. subtilis components of the SRP-like secretory pathway have been found. The Small Cytoplasmic RNA (scRNA) was shown to have a functional relationship with the human SRP 7S RNA and the E. coli 4.5S RNA (Nakamura et al., 1992). The B. subtilis scRNA is transcribed from the scr gene as a 354 nucleotide precursor which is then processed to a 271 nucleotide RNA at the 5xe2x80x2 and 3xe2x80x2 end (Struck et al., 1989), which is similar to its eukaryotic homologue (300 nucleotides) but much larger than the E. coli 4.5S RNA (114 nucleotides). Also the secundary structure of the scRNA is very similar to the eukaryotic SRP 7S RNA, lacking only the domain III (Struck and Erdmann, 1990). This is in contrast to the other eubacterial SRP-like RNAs, which only fold into a single hairpin corresponding to domain IV (Poritz et al., 1988). Therefore the B. subtilis scRNA is both in size and secundary structure an intermediate between prokaryotic and eukaryotic SRP-like RNA.
Besides the scr gene another gene encoding a SRP constituent has been isolated from B. subtilis. The ffh gene was found to encode the Ffh protein which shows homology to both the E. coli and eukaryotic SRP54 protein (Honda et al., 1993).
It is not unlikely that chaperones or members of the SRP-like secretion pathway may become a rate-limiting step in the secretion pathway, the result of which being that the majority of the heterologous protein expressed will aggregate or fold prematurely. This effect could be the reason why attempts to secrete heterologous proteins in high amounts from Gram-positive micro-organisms, in particular B. subtilis and (closely) related micro-organisms have met with little success. Overexpression of particular members of the B. subtilis secretion machinery, especially of chaperone-like proteins which are the rate-limiting step in the secretion pathway would solve this problem. It is to be understood that the terms xe2x80x9cchaperonexe2x80x9d and xe2x80x9csecretion factorxe2x80x9d are not completely clearly defined. Both groups of proteins will at least overlap and in some cases may be identical. Because the mechanism of action of these proteins is not yet clearly understood, both terms will be used interchangeably herein.
The invention thus provides a proteinaceous substance comprising at least a functional part of a chaperone-like protein expressed by Gram-positive bacteria encoded by the ftsY gene of said bacteria, a representative of said gene being defined by the sequence of seq. ID no. 7.
When Gram-positive bacteria, especially Bacillus species and in particular Bacillus subtilis and its closely related organisms are provided with this proteinaceous substance, of which the functionality is defined as being able to recognize a protein of interest to be secreted and lead it into the secretory pathway, they will have an enhanced capability of secreting proteins. It is very likely that proteins to be secreted must have a signal sequence, which may be their own signal sequence, or a signal sequence of a homologous protein of the host bacteria or a signal sequence homologous to the micro-organism from which the proteinaceous substance, i.e. the secretion factor according to the invention is derived.
The protein of interest may be any protein which up until now has been considered for expression in prokaryotes, as long as it can be provided or has of its own a signal sequence which render it suitable for secretion in a Gram-positive host. Of course it must also be able to be recognized (if possibly not very efficiently) and lead into the secretory pathway by the chaperone-like proteins according to the invention. It may not be the case that the chaperone-like proteins would be capable of recognizing and leading into secretion each and every protein by itself. Other secretion factors may become the rate-limiting step, if the presently invented secretion factor is provided in sufficient quantities. In that case it is preferred to also provide the hosts with the secretion factors which may become the rate-limiting step in sufficient quantities also. Since we believe that there is a SRP-like route in Bacillus species and other gram-positive bacteria, it would be advantageous to provide the micro-organism with enhanced amounts of FtsY, the 7S scRNA and Ffh.
The protein of interest may be either homologous or heterologous to the host. In the first case overexpression should be read as expression above normal levels in said host. In the latter case basically any expression is of course overexpression.
The proteinaceous substance according to the invention, which for convenience will often be referred to as the chaperone, secretion factor or the chaperone-like protein, may be homologous to the host, which is preferred, but it may also be heterologous to the host, as long as it is compatible with the secretion machinery of the host. It stands to reason that this will be most likely in closely related organisms. Thus in the case of a Bacillus subtilis secretion factor, it would be preferred to use it in a Bacillus.
The sequence being depicted as giving a representative of a sequence encoding a chaperone-like protein according to the invention is given in order to enable the person skilled in the art to find homologous sequences which encode similar or functionally the same chaperone-like proteins in other Gram-positive bacteria, in particular of other Bacillus species. Given the general level of skill in the art, it will be routine work to prepare for instance primers based on the given sequence and to screen for other homologous sequences encoding said chaperone-like proteins. These chaperone-like proteins from other related organisms should therefore be considered as part of the present invention. Their DNA and/or amino acid sequences usually will be quite homologous. As a rule the homology will be greater then 70% overall, in particular homologies of greater than 85% overall are to be expected. It is understood that all homologous genes which can hybridize with the sequence depicted in seq. ID no. 7 and which encode a protein of essentially the same structure or function are comprised in this invention. The following equation, which has been derived from analyzing the influence of different factors on hybrid stability:
Tm=81+16.6 (log10 Ci)+0.4 (% G+C)xe2x88x92600/nxe2x88x921.5% mismatch (Ausubel et al., supra) where
n=length of the shortest chain of the probe
Ci=ionic strength (M)
G+C=base composition,
can be used to determine which level of homology can be detected using DNAxe2x80x94DNA hybridisation techniques.
Therefore the term xe2x80x9cessentially of a structurexe2x80x9d is intended to embrace sequences which can include conservative mutations, where the sequence encodes the same amino acid, but which may differ by up to 35% in the DNA sequence according to the above equation, more typically by up to 10%. It is not always necessary to have a complete chaperone-like protein to perform the functions of recognizing the protein to be secreted and leading said protein into the secretion pathway. Where possible the hosts may thus also be provided with functional parts of said secretion factor. It is also possible and even likely that association with non-protein material such as the 7S RNA may occur when the secretion factor performs its functions. The term proteinaceous substance is chosen to include such associations. It is by now well known in the art that mutations in proteins may lead to higher activity, longer half-lives, better stability of the mutated protein. Such derivatives of the secretion factors according to the invention are also part thereof, since given the information presented herein, it is routine work to find weak spots, or other sites interesting for mutation in the secretion factors according to the invention and making site-directed mutations.
A preferred embodiment of the present invention is a proteinaceous substance which is a chaperone-like protein which is at least partly encoded by the ftsY gene of a Bacillus species. Bacillus species are highly preferred organisms to express genes of interest in and a lot of developmental and production experience is available. As stated before however, there has always been a problem with secretion of especially heterologous proteins from Bacillus. This problem may in many cases be solved by providing Bacillus organisms with chaperone-like proteins from other related species, but it will be clear that chances of a good functional secretion factor in Bacillus, including recognition of the heterologous protein are highest using a chaperone-like protein which is derived from a secretion factor in a Bacillus species. Of special interest as a chaperonelike protein is a proteinaceous substance which is at least partly encoded by the ftsY gene of Bacillus subtilis or another Bacillus species. These proteinaceous substances are very likely to be analogues of the eukaryotic docking protein (or SRP receptor) as is the case with products derived from the E. coli ftsY gene. Lack of sufficient amounts of this chaperone-like protein will definitely have a great influence on the capability of host micro-organisms to secrete any proteins, let alone heterologous proteins. At present we believe that heterologous protein may be predominantly secreted using the SRP-like route, i.e. by binding to Ffh, the 7s RNA and the FtsY, whereas homologous proteins use the general secretion pathway. It is also clear that if a heterologous protein to be secreted is provided with a signal homologous or very closely related to a signal present in Bacillus secretory proteins that the presence of a sufficient amount of the secretion factor which normally has the function of recognizing such a signal will lead to enhanced secretion.
The preferred method of providing a Gram-positive bacteria, in particular a Bacillus species with the possibility of expressing sufficient amounts of the chaperone-like proteins (and for instance scRNA) according to the invention is of course by providing said micro-organism with the genetic information to overexpress said chaperone-like protein. The invention therefor also provides a recombinant DNA molecule comprising at least a part of an ftsY gene encoding a chaperone-like protein of Gram-positive bacteria, said part encoding at least a functional part of said chaperone-like protein, whereby a representative of said gene has the sequence of seq. ID no. 7.
As is true for the proteinaceous substances of the invention the given sequence is given for the reason of enabling the skilled person to find homologous sequences encoding similar secretion factors. Variants may exist within Bacillus species and other Gram-positive bacteria. It will also enable the person skilled in the art to construe silent mutations, to construe beneficial mutations or mutations having no effect on the activity of the chaperone resulting from expression. For different species codon preference may de different, degeneracy may be accounted for. All these modifications should be considered to be within the scope of the present invention. To define which genes still belong to the invention can really only be done by their functionality. If they encode a substance which has the same activity (in kind, not in amount) as the presently invented chaperone-like proteins then the gene (or the recombinant DNA molecule) should be considered to belong to the present invention, if the molecule is derived from a Gram-positive bacteria, in particular a Bacillus species. Usually this will coincide with a rather high degree of homology for instance of 70-95% overall.
A further preferred embodiment of the present invention is of course a gene or a recombinant DNA molecule comprising at least a part of the ftsY gene of a Bacillus species. The main reason for this preference is of course that Bacillus species are well known production organisms in which for reasons already mentioned it would be helpful to provide an autologous (sometimes also called homologous) chaperone-like protein. The most preferred chaperone-like protein at the present time is the one encoded by a recombinant DNA molecule comprising at least a part of the ftsY gene of Bacillus subtilis. 
For easy transfer of the genetic information of the secretion factors according to the invention it is preferred to provide the recombinant DNA molecule as a vector. The invention thus also provides a recombinant vector comprising a recombinant DNA molecule as disclosed above and suitable regulatory elements for replication and/or expression. The nature and kind of such a vector is not important, as long as it is capable of transferring the wanted genetic information into the desired micro-organism and preferably being capable of replicating or being replicated in such micro-organism. They may comprise many additional advantageous features such as marker genes, restriction sites, etc. Chromosonal integration of (part of) the gene according to the secretion factor is also comprised within this invention. It would of course be advantageous to only have to transfer a micro-organism with one vector. Preferably the invention provides a recombinant vector as described above further comprising a gene encoding a protein of interest to be secreted.
The invention further provides micro-organisms which have been provided with the genetic information to encode a chaperone-like according to the invention by whatever method. The invention thus includes a cell derived from a Gram-positive host cell comprising a recombinant DNA molecule or a vector as defined herein before. Preferably the cell is derived from a Bacillus species.
In a further preferred embodiment the cell has also been provided with the ability to overexpress either or both 7S scRNA and Ffh in a similar manner as it has been provided with the (over)expression of FtsY.
In a further preferred embodiment the cell also has been provided with the ability to overexpress a homologous protein or to express a heterologous protein. A suitable way to arrive at such a cell is providing it with the genetic information for said protein of interest, leading to a cell comprising a vector having the genetic information encoding a chaperone-like protein according to the invention, further comprising a vector comprising a gene encoding a protein of interest to be secreted. All methods leading to the products of the invention are of course also part of this invention. In particular important are the methods leading to the enhanced production of proteins secreted in the culture medium. The invention thus also includes a method for enhancing the secretion of a protein of interest from a Gram-positive micro-organism, comprising the steps of providing said micro-organism with the possibility to over express the protein of interest, providing the micro-organism with the possibility of overexpressing a proteinaceous substance according to the invention, and culturing said micro-organism under suitable conditions.
Preferably the possibility to overexpress the protein of interest is provided by a vector as disclosed herein before and the possibility to overexpress a proteinaceous substance according to the invention is also provided by a vector as disclosed hereinabove.
The invention will now be further illustrated in the following detailed description and the examples.